Methods of gene assembly and their use in dna data storage

ABSTRACT

A system for DNA gene assembly that utilizes a DNA symbol library and a DNA linker library. The symbol library has a number of DNA symbols each having a first overhanging end and a second overhanging end different than and non-complimentary to the first end, the first and second ends being the same nucleotides for each DNA symbol. The linker library has pairs of DNA linkers, a first linker of a pair having a first overhanging end and a second overhanging end and a second linker of the pair having a first overhanging end and a second overhanging end, the first end of the first linker being the same nucleotides for each first linker and the second end of the second linker being the same nucleotides for each second linker, wherein the second end of the first linker and the first end of the second linker have complementary nucleotides. The first linker joins to the first end of a DNA symbol and the second linker joins to the second end of another DNA symbol.

CROSS-REFERENCE

This application claims priority to U.S. Provisional application No.62/889,400 filed Aug. 20, 2019 and titled “DNA Storage WriteArchitecture,” which is incorporated herein by reference for allpurposes.

This application incorporates by reference the nucleotide sequences inthe ASCII text file titled “STL074690_Sequence_Listing_S25.txt,” thedate of creation of this ASCII text file being Jul. 16, 2020, and thesize of the ASCII text file in bytes being 5 KB, the content of which isincorporated by reference, in its entirety, into this application. TheASCII text file refers to the sequences shown in the figures,particularly, in FIGS. 1A and B, FIG. 2, FIG. 5C, FIG. 7C, and FIGS.10A, 10B and 10C, where “A” refers to adenine, “G” refers to guanine,“C” refers to cytosine, and “T” refers to thymine. No new matter isbeing added to this application by addition of these sequence listings.

BACKGROUND

There is always a desire for more data storage and increased writing toand reading from that storage.

DNA is an emerging technology for data storage. Current methods assertthat a DNA strand or gene, to store 5 KB of data, can be written in 14days. Comparatively, magnetic disk drives and magnetic tapes both canwrite 1 TByte in about an hour. A single DNA base pair location canstore 2 bits; thus, 4000 Giga-base pairs would need to be stored in anhour to match the capabilities of a single disk drive or tape. Althoughcurrent technology is believed to be capable of writing 15 base pairs anhour, there needs to be an 8 to 9 order of magnitude improvement inorder for DNA data storage to be viable.

SUMMARY

This disclosure is directed to methods of building DNA strands, orgenes, at a high rate that are suitable for data storage. The methodsinclude assigning a bit pattern to each nucleotide and utilizinglibraries of pre-prepared oligos that are combined to form the desiredDNA gene, encoding the desired data.

One particular implementation described herein is a system for DNAsynthesis. The system has a DNA symbol library comprising a number ofDNA symbols each comprising a number of nucleotide pairs, the number ofDNA symbols being 4{circumflex over ( )}(the number of nucleotidepairs), each DNA symbol having a first overhanging end and a secondoverhanging end different than and non-complimentary to the firstoverhanging end, the first overhanging end and the second overhangingend being the same nucleotides for each DNA symbol. The system also hasa DNA linker library comprising pairs of DNA linkers each comprisingnucleotide pairs, a first linker of a pair having a first overhangingend and a second overhanging end and a second linker of the pair havinga first overhanging end and a second overhanging end, the firstoverhanging end of the first linker being the same nucleotides for eachfirst linker and the second overhanging end of the second linker beingthe same nucleotides for each second linker, wherein the secondoverhanging end of the first linker and the first overhanging end of thesecond linker have complementary nucleotides. The first linker of a pairis adapted to join to the first overhanging end of a DNA symbol and thesecond linker of the pair is adapted to join to the second overhangingend of another DNA symbol. In some implementations, the DNA linkerlibrary also has DNA linkers having a non-overhanging end. Additionallyor alternately, in some implementations, the first overhanging end foreach of the DNA symbols in the DNA symbol library is the same, and thesecond overhanging end for each of the DNA symbols in the DNA symbollibrary is the same.

One particular implementation described herein is a method of making aDNA gene. The method includes providing a DNA symbol library comprisinga number of DNA symbols each having a first overhanging end and a secondoverhanging end different than and non-complimentary to the firstoverhanging end, the first overhanging end and the second overhangingend being the same nucleotides for each DNA symbol, and providing a DNAlinker library comprising pairs of DNA linkers each comprisingnucleotide pairs, a first linker of a pair having a first overhangingend and a second overhanging end and a second linker of the pair havinga first overhanging end and a second overhanging end, the firstoverhanging end of the first linker being the same nucleotides for eachfirst linker and the second overhanging end of the second linker beingthe same nucleotides for each second linker, wherein the secondoverhanging end of the first linker and the first overhanging end of thesecond linker have complementary nucleotides. The method also includes,simultaneously, linking a first DNA symbol to a first first linker andto a first second linker, the first and second linkers from a pair oflinkers or from different pairs of linkers, the first overhanging end ofthe first symbol linking to the first first linker and the secondoverhanging end of the first symbol linking to the first second linkerto form a first oligo; linking a second DNA symbol to a second firstlinker and to a second second linker, the first and second linkers froma pair of linkers or from different pairs of linkers, the firstoverhanging end of the second symbol linking to the second first linkerand the second overhanging end of the second symbol linking to thesecond second linker to form a second oligo; and linking a third DNAsymbol to a third first linker and to a third second linker, the firstand second linkers from a pair of linkers or from different pairs oflinkers, the first overhanging end of the third symbol linking to thethird first linker and the second overhanging end of the third symbollinking to the third second linker to form a third oligo. The methodfurther includes linking the first oligo, the second oligo and the thirdoligo to form a DNA gene.

Other systems and methods are also described herein.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter. These andvarious other features and advantages will be apparent from a reading ofthe following detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The described technology is best understood from the following DetailedDescription describing various implementations read in connection withthe accompanying drawing.

FIG. 1A is a schematic rendering of two DNA oligos; FIG. 1B is aschematic rendering of the two DNA oligos having overhanging ends; andFIG. 1C is a schematic rendering of the two DNA oligos havingoverhanging ends joined.

FIG. 2 is a schematic rendering of two DNA oligos both having the sameoverhanging ends, the DNA oligos being symbols from an example symbollibrary.

FIG. 3A is a schematic rendering of a DNA oligo having two overhangingends, the oligo being a linker from an example linker library; and FIG.3B is a schematic rendering of two DNA oligos having one overhanging endand one terminating end, the DNA oligos being linkers from an examplelinker library.

FIG. 4 is a schematic rendering of four example pairs of linkers.

FIG. 5A is a schematic rendering of three oligo symbols, a first step ina method of making a data storage gene; FIG. 5B is schematic renderingof a second step of joining the three symbols each with two linkers, thesecond step in the method; and FIG. 5C is a schematic rendering of athird step of the method of making a data storage gene.

FIG. 6 is a schematic rendering of a data storage gene annotated to showvarious portions thereof.

FIG. 7A is a schematic rendering of six oligo symbols, a first step in amethod of making a data storage gene; FIG. 7B is schematic rendering ofa second step of joining the six symbols each with two linkers, thesecond step in the method; FIG. 7C is a schematic rendering of thejoined symbols from FIG. 7B, a third step of the method of making a datastorage gene; and FIG. 7D is a schematic rendering of symbols of FIG. 7Cjoined to form the data storage gene, a fourth step of the method.

FIG. 8A is a schematic diagram of a lab-on-a-chip showing a step in amethod of making a data storage gene; and FIG. 8B is a schematic diagramof the lab-on-a-chip showing another step in the method.

FIG. 9 is a schematic diagram of a lab-on-a-chip showing a PCR process.

FIG. 10A is a schematic rendering of a first portion of a PCR processutilizing the same primer; FIG. 10B is a schematic rendering of a secondportion of the PCR process; and FIG. 10C is a schematic rendering of aportion of an assembly process.

DETAILED DESCRIPTION

As indicated above, various methods of building DNA strands or genes ata high rate are provided herein. The methods include utilizing librariesof pre-prepared oligos and mass parallelization to form the desired DNAstructure or gene. If the gene is to be used as a data storage gene, themethods include assigning a bit pattern (e.g., 00, 01, 10, 11) to eachnucleotide (A, C, G, T), thus providing a gene encoding the desireddata. It is noted that the methods described herein are directed tosynthesizing a data storage gene, however the same methods areapplicable to other applications that warrant DNA synthesis.

In the following description, reference is made to the accompanyingdrawing that forms a part hereof and in which is shown by way ofillustration at least one specific implementation. The followingdescription provides additional specific implementations. It is to beunderstood that other implementations are contemplated and may be madewithout departing from the scope or spirit of the present disclosure.The following detailed description, therefore, is not to be taken in alimiting sense. While the present disclosure is not so limited, anappreciation of various aspects of the disclosure will be gained througha discussion of the examples, including the figures, provided below. Insome instances, a reference numeral may have an associated sub-labelconsisting of a lower-case letter to denote one of multiple similarcomponents. When reference is made to a reference numeral withoutspecification of a sub-label, the reference is intended to refer to allsuch multiple similar components.

As indicated above, for a data storage gene, each nucleotide is assigneda bit pattern. In one example, A=00, C=10, G=01, and T=11. Multiplenucleotides form an oligo, and multiple oligos can be combined toeventually form a gene.

In accordance with the system described herein, multiple oligos aregrouped in a library. An example of an oligo library is provided inTable 1, which lists pairs of nucleotides and a corresponding binarypattern.

TABLE 1 DNA Oligo Binary AA 0000 AG 0001 AC 0010 AT 0011 GA 0100 GG 0101GC 0110 GT 0111 CA 1000 CG 1001 CC 1010 CT 1011 TA 1100 TG 1101 TC 1110TT 1111

Using the example in Table 1 above, AA is 0000; the two base pair oligostores 4 bits. As the oligo strand lengthens, more bits, bytes and datacan be stored. For example, an oligo that is 8 base pairs long stores 16bits, or 2 bytes. Using the example in Table 1, an oligo AATTAGTC is0000111100011110, storing two bytes. It is noted that the example inTable 1 is an example of a primitive case and other bit mappings arepossible where both the mapping and number of nucleotides per bit aredifferent.

As indicated above, the system described herein utilizes libraries ofoligos to synthesize DNA strands or genes. The system includes a firstlibrary of oligos that are referred to herein as “symbols” and a secondlibrary of oligos that are referred to herein as “linkers.” In general,when a symbol is used in synthesizing a data storage gene, the term“symbol” is used to represent an oligo that has a bit pattern.Additional details regarding symbols and linkers are provided below.

As seen from above, longer chain oligos (symbols and/or linkers) encodemore data. Longer chains, however, typically require longer synthesistime. To decrease the time to synthesize longer chains, larger startingoligos can be used in the libraries.

For example, if the library has symbols that are 8 base pairs long, thesystem can store 16 bits per symbol. Having a DNA symbol library withlarger symbols speeds up the synthesis time, but the number of symbolsmay not scale well. For symbols that are 8 base pairs long, the systemwould have 65,536 unique symbols in the library. For symbols that are 9base pairs long, the system would have 262,144 unique symbols in thelibrary. For symbols that are 10 base pairs long, the system would have1,048,576 unique symbols. As shown in Table 2, the symbol library sizeis 4 to the power of the base pairs; i.e., the library size is4{circumflex over ( )}(base pairs per symbol).

TABLE 2 Base Pairs Number of Bits Size of per Symbol per Symbol SymbolLibrary 1 2 4 2 4 16 3 6 64 4 8 256 5 10 1024 6 12 4096 7 14 16,384 8 1665,536 9 18 262,144 10 20 1,048,576

To form a DNA strand or gene of sufficient length to store usableamounts of data, multiple DNA symbols (i.e., at least two, often atleast ten, more often at least twenty) from the library are combined. Tocontrol the connection of the symbols to obtain the desired nucleotidesequence, the symbols are provided with overhanging ends.

The overhanging ends can be generated using an isothermal buffer, anexonuclease (such as T5), a DNA ligase (such as Taq) and a DNApolymerase (e.g., a Gibson recipe). With such a procedure, a number ofbases from the 5′ ends of the symbol (oligo) are removed, creating theoverhanging ends. The overhanging ends are complementary pairs; onlyends which are complementary will combine when the symbols are combined.FIGS. 1A, 1B and 1C illustrate removal of the ends to provide hangingends and then combination of two such symbols.

In FIG. 1A, a first symbol precursor 10 a and a second symbol precursor10 b are shown. Each of these symbol precursors 10 is a DNA fragment, oroligo, formed of complementary nucleotide pairs. In the particularexample shown, each symbol precursor 10 is 20 pairs; other examples ofsymbol precursors can be shorter or longer.

In FIG. 1B, the two symbol precursors are now shown as a first symbol100 a and a second symbol 100 b, each having nucleotides removedtherefrom to form an overhanging end at each end. Specifically, thefirst symbol 100 a has a first hanging end 102 a and an opposite secondhanging end 104 a, and the second symbol 100 b has a first hanging end102 b and an opposite second hanging end 104 b. In the particularexample shown, each overhanging end is three nucleotides; other examplesof hanging ends can be shorter or longer, in most implementationshowever, longer. It is these symbols 100, plus many others, that formthe symbol library.

In FIG. 1C, the two symbols 100 from FIG. 1B are shown joined, resultingin a longer, combined symbol or oligo 1000; for ease, a delineationbetween the two symbols 100 is shown in the oligo 1000. In thisschematic, the exposed second end 104 a of the first symbol 100 a is thecomplement of the exposed first end 102 b of the second symbol 100 b,thus, the ends 104 a, 102 b join, resulting in the larger symbol 1000.

In the example shown in FIG. 1B, the hanging end 102 is not the same asthe hanging end 104 for each symbol 100, nor is the first hanging end102 a of the first symbol 100 a the same as or complimentary to thefirst hanging end 102 b of the second symbol 100 b, nor is the secondhanging end 104 a of the first symbol 100 a the same as norcomplementary to the second hanging end 104 b of the second symbol 100b. The second hanging end 104 a of the first symbol 100 a is, however,complementary to the first hanging end 102 b of the second symbol 100 b,in this example. In alternate implementations, the symbols in the symbollibrary are designed to all have the same overhanging first end and thesame overhanging second end. FIG. 2 shows two examples of symbols 200,as symbol 200 a and symbol 200 b, from a 16-bit symbol library, whichhave overhanging TT and GG ends (underlined in the figure). In theparticular example shown, each overhanging end is two nucleotides; otherexamples of hanging ends can be shorter or longer, in mostimplementations however, longer. Further, in other examples, theoverhanging ends could be any nucleotides in any sequence, e.g., AA, AC,TCG, etc., as long as the overhanging ends are not complimentary to eachother.

By having all the oligos in the symbol library have the same beginningand same end, the same PCR (polymerase chain reaction) chemistry can beused to amplify and/or replenish the inventory in the library. Becausethe ends are the same, the same two primers can be used for every symbolin the PCR process. Additional details regarding replenishing theinventory are provided below.

By having the hanging ends being the same for all the symbols 200, thesymbols 200 cannot join, as they did in the example shown in FIGS. 1Band 1C. Thus, in accordance with this disclosure, a linker library isprovided, which is a collection of “linking” oligos that will attach tothe first end and to the second end of all the symbols in the symbollibrary, thus providing a controlled connection mechanism for thesymbols. The linkers are oligos having at least one overhanging endcomplementary to an overhanging end of the symbol; the linker oligos canbe shorter than the symbol oligos. For example, if the overhanging endsfor all the symbols 200 are TT and GG, then all the linkers have atleast one overhanging end, either AA or CC, complementary to anoverhanging end of the symbol; the other end of the linker may be anynucleotide sequence and overhanging or not, pursuant to the discussionbelow regarding FIG. 4. FIG. 3A illustrates an example linker 300 havingtwo overhanging ends CC and AT (shown underlined); these ends, and thusthe linker 300, would join to a symbol having a GG end of to a symbolhaving a TA end. With these complementary linkers, the symbols assemblein the correct order to form the final data storage gene.

As used and described herein, a DNA storage gene is a collection of DNAsymbols connected by linkers. In some implementations only the term“gene” is used to refer to the DNA storage gene.

In order to obtain the correct length of the resulting data storage geneand also the correct assembly order of the symbols, the linker libraryincludes linkers having terminating or non-overhanging ends. FIG. 3Bshows two linkers 310 a, 310 b, each having one overhanging end (shownunderlined in the figure) and one terminating or non-overhanging end.Two linkers 310, each having a terminating end, will cap a chain ofassembled symbols, with one linker 310 at each end of the symbols, andwill thus terminate the data storage gene. In the shown example of FIG.3B, for the linker 310 a, the overhanging AA end will engage with a TToverhanging end of a symbol and the terminating end of the linker 310 awill terminate the gene by not allowing joining to a further symbol orlinker at that end. Similarly, for the linker 310 b, the overhanging CCend will engage with an overhanging GG end of a symbol and theterminating end of the linker 310 b will terminate the other end of thegene.

The linkers 300 having two overhanging ends can be provided as pairs, sothat at least one of the overhanging ends of each linker iscomplementary to an overhanging end of the other linker. FIG. 4 providesfour examples of linker pairs 400 a, 400 b, 400 c, 400 d. Each of thesepairs 400 has two linkers, a first linker 402 and a second linker 404,that can be connected to each other, in this implementation, in only oneconfiguration. In the particular example of pairs 400 a, 400 b, 400 c,400 d shown, each of the first linkers 402 a, 402 b, 402 c, 402 d has anoverhanging CC end and an opposite overhanging end of varyingnucleotides (AT for the linker 402 a, AC for the linker 402 b, AG forthe linker 402 c, GA for the linker 402 d), and each of the secondlinkers 404 a, 404 b, 404 c, 404 d has an overhanging AA end and anopposite overhanging end of varying nucleotides (TA for the linker 404a, TG for the linker 404 b, TC for the linker 404 c, CT for the linker404 d) that are complementary to the varying end of the first linkers402. The overhanging CC end of these first linkers 402 will join to theoverhanging GG end of the symbols 200 (of FIG. 2) and the overhanging AAof the second linkers 404 end will join to the overhanging TT end of thesymbols 200 (of FIG. 2).

Although only four linker pairs 400 are shown in FIG. 4, several otherpairs of linkers are possible. It is noted that for this example, alinker having an overhanging CC end and an opposite overhanging AA endis excluded because it will cause unwanted links.

With the library of symbols and the library of linkers, long strands orgenes can be made, such as for data storage. FIGS. 5A, 5B, 5C show stepsfor an example method using linkers and symbols to form a storage gene.

In FIG. 5A, three symbols 500 from the symbol library are shown assymbols 500 a, 500 b, 500 c. Each of the symbols 500 has two overhangingends, one end being TT and the other being GG; because of these ends,the symbols 500 will not join to each other.

In FIG. 5B, the three symbols 500 are individually combined with twolinkers from the linker library, particularly, a first linker 502 and asecond linker 504. The two linkers 502, 504 may be from the same pair(e.g., of FIG. 4) or may be from different pairs. As seen, each firstlinker 502 a, 502 b, 502 c has a CC overhanging end and a second endthat is an overhanging end (for the linker 502 a, 502 b) or aterminating end (for linker 502 c). Each second linker 504 a, 504 b, 504c has an AA overhanging end and a second end that is an overhanging end(for linker 504 b, 504 c) or a terminating end (for linker 504 a). Thesymbol 500 and the two linkers 502, 504 combine to form a longer, oligo506 (specifically, the symbol 500 a combines with the linkers 502 a, 504a to form oligo 506 a; the symbol 500 b combines with the linkers 502 b,504 b to form oligo 506 b; and the symbol 500 c combines with thelinkers 502 c, 504 c to form oligo 506 c). The symbol 500 may combinewith the two linkers 502, 504 simultaneously or sequentially; that is,the two linkers 502, 504 may combine with the symbol 500 at the sametime, or one may combine before the other. Although only three reactionsare shown progressing in parallel in this example, it is understood thatany number of reactions could simultaneously occur, thus increasing therate of building the final data storage gene.

In FIG. 5C, the oligos 506 from FIG. 5B are combined all together toform a storage gene 508. Because of the various overhanging ends, theoligo 506 a, oligo 506 b, and oligo 506 c will link in the correct orderto form the storage gene 508, and because of the terminating ends, nofurther linking on to the storage gene 508 can occur.

The previous discussion has provided an example utilizing a library ofsymbols (having overhanging ends) and a library of paired linkers toform a DNA gene or strand with the nucleotides arranged in the desiredorder. Utilizing multiple symbols and multiple linkers, all of which arepredetermined oligos, and utilizing parallel reactions, the synthesisrate of the final gene is greatly improved compared to a de novo genesynthesis where each base pair is added one at a time.

In one particular implementation, the methods of this disclosure utilizea 16-bit symbol library having 65,536 unique DNA symbols (oligos) and alinker library having 17 unique DNA linkers (oligos) having two centralbase pairs. Such as system can readily create a data storage gene thatis 15 DNA symbols long, storing 30 bytes (140 bits) using 120 basepairs. Each symbol is combined with corresponding linkers (e.g., asshown in FIG. 5B); multiple combinations can be done in parallel. Theresulting oligos 506 are then mixed to form the DNA data storage gene ina second step (e.g., as shown in FIG. 5C). It is noted that althoughthis is shown as a two-step method, there may be multiple chemistrysteps per step.

The rate of synthesis of the gene depends on the number of nucleotidepairs in the symbols and the linkers. If the linkers have three basepairs, the system can combine 63 symbols at one time to create a 126byte data storage gene that requires two steps. If the linkers have fivebase pairs, the system can combine 1023 symbols at one time to create a2048 byte data storage gene that requires two steps. Thus, the linkerlibrary provides a mechanism for readily combining the symbols in thedesired order to form the data storage gene.

Additionally, the linkers can provide timing and sequence information tothe data storage gene. The linkers provide a repetitive pattern at knownpositions in the data storage gene, as seen in FIG. 6.

In FIG. 6, a data storage gene 600, formed from symbols 610(specifically, symbols 610 a, 610 b, 610 c) linked via linkers (notcalled out in FIG. 6), is shown. The storage gene 600 has a unique startsequence 601 at a first end and a unique stop sequence 602 at the secondend, both provided by terminated ends on a linker. The linkers providerepeating patterns, in this example a first repeating pattern 602 and asecond repeating pattern 604 (both having two occurrences, as pattern602 a, 602 b and pattern 604 a, 604 b). These repeating patterns 602,604 are at the ends of the linker and can be used for timing recovery.The linkers also provide unique known patterns 606, 608 at the center ofthe linker. These unique known patterns 606, 608 can be used as addressmarks in the gene 600. Thus, each linker provides a first repeatingpattern 602 (which repeats in all the linkers), a second repeatingpattern 604 (which repeats in all the linkers), and a known pattern 606or 608. The patterns 602, 604, 606, 608, as well as the unique startsequence 601 and the unique stop sequence 602, can additionally be usedto identify partial fragments.

The linker library can be designed to reduce the number of linker oligosneeded. In such a manner, one linker can be used for multipleconnections. In general, the size of the linker library can be limitedby having additional steps in the synthesis method.

FIG. 7A through 7D show example steps for making a data storage genewith recycling of the linker library. In FIG. 7A, six symbols 700 a, 700b, 700 c, 700 d, 700 e, 700 f are shown. Each of these symbols 700 hasoverhanging ends that are the same for each symbol 700.

In FIG. 7B, the six symbols 700 are individually combined two linkersfrom the linker library, particularly, a first linker 702 and a secondlinker 704. The two linkers 702, 704 may be from the same pair (e.g., ofFIG. 4) or may be from different pairs. As seen, each first linker 702has a CC overhanging end. Linkers 702 a, 702 b, 702 c, 702 d and 702 ehave a second end that is an overhanging end of various nucleotides,except that the ends are the same for linkers 702 a and 702 d, forlinkers 702 b and 702 e. The linker 702 f has a truncated or terminatingsecond end. Each second linker 704 has an AA overhanging end. Linkers704 b, 704 c, 704 d, 704 e and 704 f have a second end that is anoverhanging end of various nucleotides, except that the ends are thesame for linkers 704 b and 704 e, and for linkers 704 c and 704 f. Thelinker 704 a has a truncated or terminating second end.

The symbol 700 and the two linkers 702, 704 combine to form a longer,combined oligo 706 (specifically, symbol 700 a combines with linkers 702a, 704 a to form oligo 706 a; symbol 700 b combines with linkers 702 b,704 b to form oligo 706 b, etc.). Although only six reactions are shownprogressing in parallel in this example, it is understood that anynumber of reactions could simultaneously occur, thus increasing the rateof synthesis.

FIG. 7C shows intermediate oligo 708 a formed by linking combined oligo706 a, combined oligo 706 b and combined oligo 706 c (all from FIG. 7B)directly together via their overhanging ends, and intermediate oligo 708b formed by linking combined oligo 706 d, combined oligo 706 e andcombined oligo 706 f directly together via their overhanging ends. Thefirst intermediate oligo 708 a has a terminal end due to linker 704 aand the second intermediate oligo 708 b has a terminating end due tolinker 702 f.

In FIG. 7D, the intermediate oligo 708 a and intermediate oligo 708 bare combined to form a data storage gene 710, without the need to useadditional linkers due to the complementary overhanging ends.

Depending on the terminal ends of the symbols and the linkers,additional step(s) may be included combining an oligo (e.g., anintermediate oligo) with a pair of linkers to form yet a longer oligo,which is then joined in a subsequent step, such as in FIG. 7D.

Summarized, for a gene that is 64 symbols long, the following methodscan be used to synthesize the gene.

Method #1: Step 1: mix 64 oligo symbols with their corresponding linkeroligos from the linker library which contains 64 unique pairs oflinkers. Step 2: mix all 64 oligos to form the gene.

Method #2: Step 1: mix 16 oligo symbols with their corresponding linkeroligos from the linker library which contains 16 unique pairs oflinkers. Step 2: mix each of the oligos from step 1 together to form a16 symbol oligo. Step 3: repeat steps 1 and 2 three more time with 32additional symbols. Step 4: after step 3, there are 4 oligos that areeach 16 symbols long; mix these individually with 4 pairs of linkers.Step 5: combine all 4 oligos from step 4 to create a gene that is 64symbols long. The repeats of step 1 and step 2 (described in step 3) canbe done in parallel.

As can be seen, Method #2 requires more steps, but also utilizes only 16linkers versus the 64 linkers for Method #1.

Similarly, for a gene that is 60 symbols long, the following methods canbe used to synthesize the gene.

Method #1: Step 1: mix 60 oligo symbols with their corresponding linkeroligos from the linker library which contains 60 unique pairs oflinkers. Step 2: mix all 60 oligos to form the gene.

Method #2: Step 1: mix 15 oligo symbols with their corresponding linkeroligos from the linker library which contains 15 unique pairs oflinkers. Step 2: mix each of the oligos from step 1 together to form a15 symbol oligo. Step 3: repeat steps 1 and 2 three more time with 30additional symbols. Step 4: after step 3, there are 4 oligos that areeach 15 symbols long; mix these individually with 4 pairs of linkers.Step 5: combine all 4 oligos from step 4 to create a gene that is 60symbols long. The repeats of step 1 and step 2 (described in step 3) canbe done in parallel.

As can be seen, Method #2 requires more steps, but also utilizes only 15linkers versus the 60 linkers for Method #1.

With such methods, the numbers of linkers in the linker library can bereduced or limited by utilizing the same overhanging ends and includingadditional steps in the synthesis method. For example, a 15 linker-pairlinker library reused twice will give a 15×15=225 symbol gene in foursteps. A 16 linker-pair linker library reused twice will give a 16×6=256symbol storage gene in four steps; at 2 bytes per symbol, the result isa 512 byte storage gene. As another example, a 64 linker-pair linkerlibrary reused twice will give a 64×64=4096 symbol storage gene in foursteps; at 2 bytes per symbol, the result is an 8192 byte storage gene.As yet another example, a 4096 linker-pair linker library reused twicewill give a 4096×4096=16,777,216 symbol storage gene in four steps; at 2bytes per symbol, the result is a 33 megabyte storage gene.

In the example provided above, the system has 65,536 unique DNA symbolsin the symbol library, each which is 16 bits on 8 base pairs.

Once a data storage gene is formed, the data stored therein, by thesequence of the nucleotides, can be read by known sequencing methods.However, during reading of the data storage gene, errors may occur. Byreading one nucleotide base incorrectly, two bit errors are obtained.For example:

-   -   Correct read: AATTAGTC translates to 00001111000110    -   Incorrect read: TATTAGTC translates to 11001111000110

To inhibit incorrect reading, an error correction can be built in to theDNA symbols. With the system described herein, extra base pairs can beadded to the symbols to create a Hamming Code; adding extra pairs to thesymbols does not increase the size of the library nor slow down thesynthesis of the data storage gene. It is noted that the extra basepairs may, however, decrease the read speed of the gene. Hamming Codesare well known in other applications, and additional details regardingsame are well known and are not provided herein.

The synthesis method described above can be implemented in any manner,e.g., utilizing various reactors, flasks, beakers, etc. The method isalso particularly suited to be done as a microfluidic lab-on-a-chipprocess.

Lab-on-a-chip is a common term for an integrated circuit (“chip”) onwhich one or several laboratory functions or chemical reactions aredone. The chip can be no more than a few square centimeters.Labs-on-a-chip handle extremely small fluid volumes (e.g., measured aspico-liters) and are often called microfluidic systems. In digitalmicrofluidics, the lab-on-a-chip has a hydrophobic “chip platform” onwhich fluid droplets (e.g., liquid droplets) can be manipulated byprecisely controlled voltage application. The platform may have a coverplate covering the fluidic area. By utilizing the feature of surfacetension of the fluid on the platform, the fluid can be precisely movedacross the platform by voltage applied to the platform, e.g., in a grid.

For the synthesis method described above, the lab-on-a-chip is operablyand fluidically connected to the symbol library, with each symbolretained in a well or other liquid storage compartment. Similarly, thelab-on-a-chip is operably and fluidically connected to the linkerlibrary, with each linker retained in a well or other storagecompartment. In some designs, there may be at least 10,000 wells for thesymbols, or at least 20,000, or at least 30,000 wells, or at least65,000 wells. Additionally or alternately, there can be at least 10wells for the linkers, or at least 15 wells, at least 30 wells, or atleast 60 wells.

Using known techniques (e.g., voltage differential on the platform), thedispensed symbols and linkers are moved on (across) the platform andmixed in the desired steps. All mixing of the oligos (e.g., symbols andlinkers) can be done on the platform or a dedicated mixing station maybe used for one or more of the joining steps, e.g., utilizing heatand/or agitation. In some implementations, the platform may include acontrollable reaction facilitator, such as a UV light source, and/or thefinal mixing station may include a voltage source, e.g., to align thecompleted gene to aid in collection.

One suitable (physical) size for a lab-on-a-chip is about 20 mm by 20mm, which is compatible to an 8 inch wafer and could have 785,000 arrayelements, each array element having controllable voltage independentlyapplied thereto. In some implementations, each well or other storagecompartment for the oligos (symbols or linkers) is 10× the size of anarray element. This would provide 66,560 wells and leave 119,000 arraysfor transport and mixing of the symbols and linkers on the platform.

A stacked or otherwise three-dimensional array of labs-on-a-chip wouldincrease density and decrease required area for the synthesis. A dropelevator could be used to provide synthesis on multiple verticallystacked levels.

A cleaning or decontamination mechanism may be included in thelab-on-the-chip to rinse, wash, or otherwise decontaminate certain orall grid locations that have had or will have a symbol or linker presentthereon. For example, an amount (e.g., drop) of cleaning solution (e.g.,hydrogen peroxide) can be applied to and moved across the platform tocleanse the platform. In one particular example, the cleaning solutioncan follow immediately behind a linker or symbol, thus cleaning anddecontaminating the surface of any oligo that may remain. In anotherparticular example, the cleaning solution can trace the path the oligowill follow.

FIGS. 8A and 8B illustrate two steps of an example synthesis method.These figures illustrate an example of a lab-on-a-chip to make a 2048byte storage gene using the methods of this disclosure.

FIGS. 8A and 8B show a lab-on-a-chip 800 with a platform working surface802 having numerous cells each configured for independently receiving avoltage. The lab 800 includes a plurality of wells 804 for the oligosymbol library, each well 804 retaining one symbol. The lab 800 alsoincludes a plurality of wells 806 for the oligo linker library, eachwell 806 retaining one linker. Although the figures show the wells 804and the wells 806 on opposite sides of the platform 802, because theremay be significantly more symbol wells 804 than linker wells 806, thewells 804, 806 may be arranged on the chip 800 in any order. To make a2048 byte gene, 65,536 symbols are present in the wells 804 and 1024linker pairs (thus, 2048 linker oligos) are present in the wells 806.The lab 800 also has a final mixing location 808 for the final mixing orsynthesis step for the data storage gene.

In a first step, partially shown in FIG. 8A, all 1024 linker pairs arecombined with their corresponding 1024 (of the 65,536) symbols on theplatform 802; for clarity of understanding and to simplify the figure,FIG. 8A shows only four combinations of three unique symbols with eightunique linkers, although all linkers and symbols may eventually becombined on the platform 802. The selected symbol is moved via voltageon the platform 802 to meet and combine with the appropriate linkers(also moved via voltage on the platform 802). In a second step, shownpartially in FIG. 8B, all 1024 drops (which have a symbol with twolinkers) are moved via voltage to the final mixing location 808 wherethey self-assemble to form the 2048 byte data storage gene; for clarityof understanding and to simplify the figure, FIG. 8B shows the fourcombinations moving to the final mixing location 808, although allcombined linkers and symbols will eventually move to the final mixinglocation 808. It is noted that a particular symbol and/or particularlinkers may be used multiple times to form the eventual gene.Additionally, a particular symbol can be combined with differentlinkers, as well as a particular symbol can be combined with differentlinkers.

The lab 800 also includes a PCR region 810 to replenish the linkerand/or symbol libraries, the PCR region 810 including wells for PCRchemicals 820 a, 820 b and a PCR station 830. Naturally, the symbols andlinkers are depleted with each synthesized storage gene. Occasionally,the symbols and linkers need to be replenished; the PCR region 810 ofthe lab 800 allows this replenishment to be done at the lab 800.

Depending on the symbols and the linkers used (particularly, theoverhanging ends of the symbols and the linkers), the same PCR chemistryset can be used for both the symbol and linker libraries. In someimplementations, only a few (e.g., one, two, three, or four) PCRchemicals are needed.

Because of the need to move numerous symbols and linkers to each other,to the final mixing location 808, and to the PCR region 810, many ofwhich are moved or moving simultaneously, numerous paths are used. Forexample, at a point in time, one hundred symbols and 200 linkers (e.g.,16 unique linker pairs, some of which are used multiple times) may bemoving on the platform 802. In most implementations, these paths are notconstrained by channels or other physical or set paths on the platform802, but movement of the fluids on the platform 802 is controlled merelyby the applied voltage. It is noted that due to the large number ofpaths needed, a very detailed and complicated traffic map may be needed.

FIG. 9 illustrates use of the PCR region to replenish a symbol. Similarto the lab 800, in FIG. 9 the lab-on-a-chip 900 has a platform workingsurface 902 having numerous cells each configured for independentlyreceiving a voltage. The lab 900 includes a plurality of wells 904 forthe oligo symbol library, a plurality of wells 906 for the oligo linkerlibrary, and a final mixing location 908. The lab 900 also includes aPCR region 910 to replenish the linker and/or symbol libraries whenneeded, the PCR region 910 including wells 902 a, 902 b for PCRchemicals and a PCR station 930.

In FIG. 9, a symbol is shown being moved from its respective well 904 tothe PCR station 930. Appropriate PCR chemicals (e.g., primers, DNApolymerase, free nucleotides) are added from the chemical wells 920 tothe station 930 to synthesize additional copies of the symbol. The lab900 can include an appropriate heating source to denature the symbol orlinker being synthesized. Additionally, the lab 900 can include anappropriate cooling source for annealing primers to the denatured symbolor linker. The PCR station 930 is configured to include all chemicalsneeded to automatically and autonomously replenish the symbols andlinkers when needed.

In a PCR process, two primers are needed for each oligo, one primer foreach end. As indicated above, by having all the oligos in the symbollibrary have the same beginning and same end (TT and GG overhangingends, in the example shown), the same PCR chemistry (i.e., the same twoprimers) can be used for all symbols in the library. In the exampleprovided above however, half of the oligos in the linker library havethe same first end and the other half of the oligos in the linkerlibrary have another same first end; the second end is different. Forthe linkers, the same PCR chemistry (i.e., the same primer) can be usedfor one end of all the linkers; only the second end of the linkers willneed a different primer.

To avoid the need for numerous primer chemistries, the oligos and theprimer can be specifically designed for each other. In the followingexample shown in FIGS. 10A, 10B and 10C, a universal primer for all DNAsymbols, linkers, and terminating ends is used for PCR amplification.

In these figures, a forward primer “PF”, and a reverse primer, “PR” arecomplimentary to the 3′ ends of each DNA oligo (the oligo being asymbol, linker, or terminating end and found at the center region ofeach oligo, further discussed below). During PCR amplification, primerPF anneals to the forward 3′ end and primer PR anneals to the reverse 3′end.

FIG. 10A and FIG. 10B show these universal primer binding sequences, PRand PF, attached to two reaction sets, one in FIG. 10A and one in FIG.10B. In FIG. 10A, the set 1000A has an oligo 1002 that contains a linker(linker 1A, depicted as L1A), an oligo 1004 that contains a terminatingend (depicted as E1), and an oligo 1006 that contains a symbol (depictedas S1). In FIG. 10B, the set 1000B has an oligo 1052 that contains alinker (linker 1B, depicted as LIB), an oligo 1054 that contains alinker (linker 2A, depicted as L2A), and an oligo 1056 that contains asymbol (depicted as S2). The nucleotides shown in bold in the oligos arenecessary internal bases and are adjacent to the linker, symbol, orterminating end.

Downstream of the forward primer PF binding region, there is arestriction enzyme cut site; in the shown example, the cut site is aBamH1 site, identified as

G/GATCC CCATG/Gin each of oligos 1002, 1004, 1006 and oligos 1052, 1054, 1056.

Upstream of the reverse primer PR binding region, there is a secondrestriction enzyme cut site; in the shown example, the cut site is aBcl1 site, identified as

T/GATCA ACTAG/Tin each of oligos 1002, 1004, 1006 and oligos 1052, 1054, 1056.

The slashes (/) indicate the locations where the restriction enzymescut.

The two cut sites, at the forward primer PF and the reverse primer PR,are different in this example but in other implementations the cut sitescan be the same.

After PCR amplification, the primer binding regions may be cut off therest of the DNA segment by the addition of the appropriate restrictionenzyme. In FIG. 10A, the oligos 1002, 1004, 1006 are cut at the Bcl1 andBamH1 sites (e.g., by a reaction that takes 5-15 minutes at 37° C.) toform a next set 1010 of oligos, specifically, the oligos 1012, 1014,1016. Similarly, in FIG. 10B, the oligos 1052, 1054, 1056 are cut at theBcl1 and BamH1 sites (e.g., by a reaction that takes 5-15 minutes at 37°C.) to form a next set 1060 of oligos, specifically, the oligos 1062,1064, 1066.

In the example provided above, the cutting reaction takes 5-15 minutesat 37° C. The reaction process may be done at any elevated temperature,e.g., 37° C. or 45° C., dependent on the particular restriction enzymeutilized. After a specified reaction time (e.g., 5-60 minutes), thereaction may be stopped by any known mechanism, including by elevatingthe temperature further for a specified time (e.g., 65° C. for 5-15minutes) or the addition of EDTA. Alternatively, if the restrictionenzyme reaction does not require a stop step, the stop step may beeliminated. Oligos 1012, 1014, 1016 in FIG. 10A and oligos 1062, 1064,1066 in FIG. 10B are the resulting DNA segments after the primers arecut off by a Bcl1 and BamH1 restriction digest.

After the primers are removed by the restriction enzyme digest, asdescribed above, the resulting DNA segments (e.g., oligos 1012, 1014,1016 and 1062, 1064, 1066) may be assembled as previously described, orthe DNA segments may be further processed.

A Gibson assembly method can be used to chew back the 5′ ends togenerate complementary overhangs. The oligos 1012, 1014, 1016 and theoligos 1062, 1064, 1066 of each of the sets 1010, 1060, respectively,can undergo a chew-back during a Gibson assembly process to arrive atthe set 1020 in FIG. 10A having oligos 1022, 1024, 1026 and the set 1070in FIG. 10B having oligos 1072, 1074, 1076.

Turning to FIG. 10C, the complimentary overhangs of the DNA segments oroligos of the sets 1020, 1070 can then be joined via Gibson assembly tofill any gaps and generate two storage gene fragments (not shown in FIG.10C). Subsequently, the two storage gene fragments may be combined in aseparate assembly reaction (e.g., Gibson assembly) to form a largerstorage gene fragment or a complete storage gene, as shown in FIG. 10C.

It is noted that although not specifically stated, between any of theassembly steps described throughout this description, any additionalsteps may be added as needed or desired, for example, a PCRamplification step, a purification step, or both. Either of theseexample steps could be performed after a Gibson assembly step.

The above specification and examples provide a complete description ofthe structure and use of exemplary implementations of the invention. Theabove description provides specific implementations. It is to beunderstood that other implementations are contemplated and may be madewithout departing from the scope or spirit of the present disclosure.The above detailed description, therefore, is not to be taken in alimiting sense. While the present disclosure is not so limited, anappreciation of various aspects of the disclosure will be gained througha discussion of the examples provided.

Unless otherwise indicated, all numbers expressing feature sizes,amounts, and physical properties are to be understood as being modifiedby the term “about,” whether or not the term “about” is immediatelypresent. Accordingly, unless indicated to the contrary, the numericalparameters set forth are approximations that can vary depending upon thedesired properties sought to be obtained by those skilled in the artutilizing the teachings disclosed herein.

As used herein, the singular forms “a”, “an”, and “the” encompassimplementations having plural referents, unless the content clearlydictates otherwise. As used in this specification and the appendedclaims, the term “or” is generally employed in its sense including“and/or” unless the content clearly dictates otherwise.

Spatially related terms, including but not limited to, “bottom,”“lower”, “top”, “upper”, “beneath”, “below”, “above”, “on top”, “on,”etc., if used herein, are utilized for ease of description to describespatial relationships of an element(s) to another. Such spatiallyrelated terms encompass different orientations of the device in additionto the particular orientations depicted in the figures and describedherein. For example, if a structure depicted in the figures is turnedover or flipped over, portions previously described as below or beneathother elements would then be above or over those other elements.

Since many implementations of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended. Furthermore, structuralfeatures of the different implementations may be combined in yet anotherimplementation without departing from the recited claims.

What is claimed is:
 1. A system for DNA synthesis, comprising: a DNAsymbol library comprising a number of DNA symbols each comprising anumber of nucleotide pairs, the number of DNA symbols being 4{circumflexover ( )}(the number of nucleotide pairs), each DNA symbol having afirst overhanging end and a second overhanging end different than andnon-complimentary to the first overhanging end, the first overhangingend and the second overhanging end being the same nucleotides for eachDNA symbol; and a DNA linker library comprising pairs of DNA linkerseach comprising nucleotide pairs, a first linker of a pair having afirst overhanging end and a second overhanging end and a second linkerof the pair having a first overhanging end and a second overhanging end,the first overhanging end of the first linker being the same nucleotidesfor each first linker and the second overhanging end of the secondlinker being the same nucleotides for each second linker, wherein thesecond overhanging end of the first linker and the first overhanging endof the second linker have complementary nucleotides, wherein the firstlinker of a pair is adapted to join to the first overhanging end of aDNA symbol and the second linker of the pair is adapted to join to thesecond overhanging end of another DNA symbol.
 2. The system of claim 1,wherein the DNA linker library further comprises DNA linkers having anon-overhanging end.
 3. The system of claim 1, wherein the DNA symbollibrary has 65,536 DNA symbols each symbol having 8 nucleotide pairs. 4.The system of claim 1, wherein the first overhanging end for each of theDNA symbols in the DNA symbol library is the same, and the secondoverhanging end for each of the DNA symbols in the DNA symbol library isthe same.
 5. A method of making a DNA gene, comprising: providing a DNAsymbol library comprising a number of DNA symbols each having a firstoverhanging end and a second overhanging end different than andnon-complimentary to the first overhanging end, the first overhangingend and the second overhanging end being the same nucleotides for eachDNA symbol; providing a DNA linker library comprising pairs of DNAlinkers each comprising nucleotide pairs, a first linker of a pairhaving a first overhanging end and a second overhanging end and a secondlinker of the pair having a first overhanging end and a secondoverhanging end, the first overhanging end of the first linker being thesame nucleotides for each first linker and the second overhanging end ofthe second linker being the same nucleotides for each second linker,wherein the second overhanging end of the first linker and the firstoverhanging end of the second linker have complementary nucleotides;simultaneously: linking a first DNA symbol to a first first linker andto a first second linker, the first and second linkers from a pair oflinkers or from different pairs of linkers, the first overhanging end ofthe first symbol linking to the first first linker and the secondoverhanging end of the first symbol linking to the first second linkerto form a first oligo; linking a second DNA symbol to a second firstlinker and to a second second linker, the first and second linkers froma pair of linkers or from different pairs of linkers, the firstoverhanging end of the second symbol linking to the second first linkerand the second overhanging end of the second symbol linking to thesecond second linker to form a second oligo; and linking a third DNAsymbol to a third first linker and to a third second linker, the firstand second linkers from a pair of linkers or from different pairs oflinkers, the first overhanging end of the third symbol linking to thethird first linker and the second overhanging end of the third symbollinking to the third second linker to form a third oligo; and linkingthe first oligo, the second oligo and the third oligo to form the DNAgene.
 6. The method of claim 5, wherein: linking the first DNA symbol tothe first first linker and to the first second linker comprisessimultaneously linking the first DNA symbol to the first first linkerand to the first second linker; linking the second DNA symbol to thesecond first linker and to the second second linker comprisessimultaneously linking the second DNA symbol to the second first linkerand to the second second linker; and linking the third DNA symbol to thethird first linker and to the third second linker comprisessimultaneously linking the third DNA symbol to the third first linkerand to the third second linker.
 7. The method of claim 5, wherein:linking the first DNA symbol to the first first linker and to the firstsecond linker comprises sequentially linking the first DNA symbol to thefirst first linker and to the first second linker; linking the secondDNA symbol to the second first linker and to the second second linkercomprises sequentially linking the second DNA symbol to the second firstlinker and to the second second linker; and linking the third DNA symbolto the third first linker and to the third second linker comprisessequentially linking the third DNA symbol to the third first linker andto the third second linker.
 8. The method of claim 5, wherein linkingthe first oligo, the second oligo and the third oligo is withoutadditional linkers.
 9. The method of claim 5, wherein linking the firstoligo, the second oligo and the third oligo is with additional firstlinkers and additional second linkers.
 10. The method of claim 5,wherein: linking the first DNA symbol to the first first linker and tothe first second linker to form the first oligo; linking the second DNAsymbol to the second first linker and to the second second linker toform the second oligo; linking the third DNA symbol to the third firstlinker and to the third second linker to form the third oligo; andlinking the first oligo, the second oligo and the third oligo to formthe DNA gene, are all done on a digital microfluidic platform.
 11. Themethod of claim 5, further comprising, simultaneously to forming thefirst oligo, the second oligo and the third oligo: linking a fourth DNAsymbol to a fourth first linker and to a fourth second linker, the firstand second linkers from a pair of linkers or from different pairs oflinkers, the first overhanging end of the fourth symbol linking to thefourth first linker and the second overhanging end of the fourth symbollinking to the fourth second linker to form a fourth oligo; linking afifth DNA symbol to a fifth first linker and to a fifth second linker,the first and second linkers from a pair of linkers or from differentpairs of linkers, the first overhanging end of the fifth symbol linkingto the fifth first linker and the second overhanging end of the fifthsymbol linking to the fifth second linker to form a fifth oligo; andlinking a sixth DNA symbol to a sixth first linker and to a sixth secondlinker, the first and second linkers from a pair of linkers or fromdifferent pairs of linkers, the first overhanging end of the sixthsymbol linking to the sixth first linker and the second overhanging endof the sixth symbol linking to the sixth second linker to form a sixtholigo; and linking the first oligo, the second oligo, the third oligo,the fourth oligo, the fifth oligo, and the sixth oligo to form the DNAgene.
 12. The method of claim 11, wherein: linking the first DNA symbolto the first first linker and to the first second linker comprisessimultaneously linking the first DNA symbol to the first first linkerand to the first second linker; linking the second DNA symbol to thesecond first linker and to the second second linker comprisessimultaneously linking the second DNA symbol to the second first linkerand to the second second linker; linking the third DNA symbol to thethird first linker and to the third second linker comprisessimultaneously linking the third DNA symbol to the third first linkerand to the third second linker; linking the fourth DNA symbol to thefourth first linker and to the fourth second linker comprisessimultaneously linking the fourth DNA symbol to the fourth first linkerand to the fourth second linker; linking the fifth DNA symbol to thefifth first linker and to the fifth second linker comprisessimultaneously linking the fifth DNA symbol to the fifth first linkerand to the fifth second linker; and linking the sixth DNA symbol to thesixth first linker and to the sixth second linker comprisessimultaneously linking the sixth DNA symbol to the sixth first linkerand to the sixth second linker.
 13. The method of claim 11, whereinlinking the first oligo, the second oligo, the third oligo, the fourtholigo, the fifth oligo, and the sixth oligo is without additionallinkers.
 14. The method of claim 11, wherein: linking the first DNAsymbol to the first first linker and to the first second linker to formthe first oligo; linking the second DNA symbol to the second firstlinker and to the second second linker to form the second oligo; linkingthe third DNA symbol to the third first linker and to the third secondlinker to form the third oligo; linking the fourth DNA symbol to thefourth first linker and to the fourth second linker to form the fourtholigo; linking the fifth DNA symbol to the fifth first linker and to thefifth second linker to form the fifth oligo; linking the sixth DNAsymbol to the sixth first linker and to the sixth second linker to formthe sixth oligo; and linking the first oligo, the second oligo, thethird oligo, the fourth oligo, the fifth oligo and the sixth oligo toform the DNA gene, are all done on a digital microfluidic platform. 15.The method of claim 5 wherein the first and second linkers are fromdifferent pairs of linkers.
 16. The method of claim 5, wherein the firstoverhanging end for each of the DNA symbols in the DNA symbol library isthe same, and the second overhanging end for each of the DNA symbols inthe DNA symbol library is the same.
 17. The method of claim 5, whereinone of the first linkers of the pairs has a terminating end and one ofthe second linkers of the pairs has a terminating end.
 18. The method ofclaim 5, wherein the number of DNA symbols each comprises a number ofnucleotide pairs, the number of DNA symbols being 4{circumflex over( )}(the number of nucleotide pairs).
 19. The method of claim 5, whereinthe DNA symbol library has 65,536 DNA symbols each symbol having 8nucleotide pairs.
 20. The method of claim 5, further comprising:assigning a two-digit binary pattern to each nucleotide.